Under-Agarose Folate Chemotaxis of <i>Dictyostelium discoideum</i> Amoebae in Permissive and Mechanically Inhibited Conditions

Laevsky, Gary; Knecht, David A.

doi:10.2144/01315rr03

Cited by 68 publications

(79 citation statements)

References 14 publications

(13 reference statements)

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“…The chemotaxis defects of pia Ϫ cells were not as strong as those for lst8 Ϫ and rip3 Ϫ cells. To further characterize the chemotaxis defects of rip3 Ϫ , pia Ϫ , and lst8 Ϫ cells, we analyzed the mutants using the underagarose assay (Laevsky and Knecht, 2001;Comer et al, 2005), in which cells migrate under a layer of agarose in a gradient of chemoattractant. Under these conditions, the lst8 Ϫ cells, as well as the rip3 Ϫ and pia Ϫ cells, do not migrate as far toward the cAMP source as wild-type cells and, most notably, do not organize into streams, a process that requires ACA activation (Kriebel et al, 2003;unpublished data).…”

Section: Dictyostelium Torc2 Regulates Cell Movement During Chemotaxismentioning

confidence: 99%

TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium

Lee

Comer

Sasaki

et al. 2005

MBoC

184

217

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Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G proteincoupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.

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Section: Dictyostelium Torc2 Regulates Cell Movement During Chemotaxismentioning

confidence: 99%

TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium

Lee

Comer

Sasaki

et al. 2005

MBoC

184

217

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“…Immotile, rounded cells were produced by placing the coverslip on ice for 10 minutes. For under-agar experiments (Laevsky and Knecht, 2001) 0.3-0.5 ml 0.6-0.7% SeaKem GTG agarose in KK 2 was added per coverglass chamber and a 2ϫ15 mm trough was cut and filled with 25 l of KK 2 containing 1-5ϫ10 4 cells. Cells crawled under the agar by random movement, or chemotaxis after doping the agar with 1 M cAMP.…”

Section: Microscopy and Image Analysismentioning

confidence: 99%

Possible roles of the endocytic cycle in cell motility

Traynor

Kay

2007

Journal of Cell Science

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Starving, highly motile Dictyostelium cells maintain an active endocytic cycle, taking up their surface about every 11 minutes. Cell motility depends on a functional NSF (N-ethylmaleimide sensitive factor) protein - also essential for endocytosis and membrane trafficking generally - and we, therefore, investigated possible ways in which the endocytic cycle might be required for cell movement. First, NSF, and presumably membrane trafficking, are not required for the initial polarization of the leading edge in a cyclic-AMP gradient. Second, we can detect no evidence for membrane flow from the leading edge, as photobleached or photoactivated marks in the plasma membrane move forward roughly in step with the leading edge, rather than backwards from it. Third, we find that the surface area of a cell - measured from confocal reconstructions - constantly fluctuates during movement as it projects pseudopodia and otherwise changes shape; increases of 20-30% can often occur over a few minutes. These fluctuations cannot be explained by reciprocal changes in filopodial surface area and they substantially exceed the 2-3% by which membranes can stretch. We propose that the endocytic cycle has a key function in motility by allowing adjustment of cell surface area to match changes in shape and that, without this function, movement is severely impaired.

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“…In standard conditions on a 2D surface under buffer, they move mainly with F-actin-driven pseudopods, but switch progressively to bleb-driven motility when faced with mechanical resistance to their movement (12). This can be conveniently applied by inducing the cells to migrate under an elastic overlay, such as agarose, which they must deform to progress (13). Blebbing is stimulated by acute treatment with the chemoattractant cyclic AMP (14), and blebs can be chemotactically orientated by cyclic-AMP gradients (11,12).…”

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confidence: 99%

How blebs and pseudopods cooperate during chemotaxis

Tyson

Zatulovskiy

Kay

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

110

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Two motors can drive extension of the leading edge of motile cells: actin polymerization and myosin-driven contraction of the cortex, producing fluid pressure and the formation of blebs. Dictyostelium cells can move with both blebs and actin-driven pseudopods at the same time, and blebs, like pseudopods, can be orientated by chemotactic gradients. Here we ask how bleb sites are selected and how the two forms of projection cooperate. We show that membrane curvature is an important, yet overlooked, factor. Dictyostelium cells were observed moving under agarose, which efficiently induces blebbing, and the dynamics of membrane deformations were analyzed. Blebs preferentially originate from negatively curved regions, generated on the flanks of either extending pseudopods or blebs themselves. This is true of cells at different developmental stages, chemotaxing to either folate or cyclic AMP and moving with both blebs and pseudopods or with blebs only. A physical model of blebbing suggests that detachment of the cell membrane is facilitated in concave areas of the cell, where membrane tension produces an outward directed force, as opposed to pulling inward in convex regions. Our findings assign a role to membrane tension in spatially coupling blebs and pseudopods, thus contributing to clustering protrusions to the cell front.C rawling cells must restrict protrusions to a limited part of their periphery if they are to move efficiently, and when these cells chemotax, the location of projections must be further controlled by the chemotactic gradient (1-3). Cellular protrusions are of two main types: those driven by actin polymerization, such as pseudopods or lamellipods, and those driven by fluid pressure, which are usually called blebs. Blebs form when the cell membrane locally detaches from the underlying cortex and is driven outward by hydrostatic pressure, created by myosin-IIdriven contraction of the cortex (4, 5). When blebs form, the cortex is left behind as an "F-actin scar," which depolymerizes, while a new actin cortex forms at the freshly exposed membrane.Blebbing is important in cells migrating in three-dimensional environments, such as during tumor invasion (6, 7), zebrafish primordial germ cell migration (8, 9), or migration of the pathogen Entamoeba histolytica in the liver (10). Dictyostelium amoebae can also move with blebs (11). In standard conditions on a 2D surface under buffer, they move mainly with F-actin-driven pseudopods, but switch progressively to bleb-driven motility when faced with mechanical resistance to their movement (12). This can be conveniently applied by inducing the cells to migrate under an elastic overlay, such as agarose, which they must deform to progress (13). Blebbing is stimulated by acute treatment with the chemoattractant cyclic AMP (14), and blebs can be chemotactically orientated by cyclic-AMP gradients (11,12).Actin-driven pseudopods are preferentially formed up-gradient by chemotaxing cells, and they can be induced on the flanks of cells by applying a steep gradient of ch...

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Under-Agarose Folate Chemotaxis of Dictyostelium discoideum Amoebae in Permissive and Mechanically Inhibited Conditions

Cited by 68 publications

References 14 publications

TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium

TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium

Possible roles of the endocytic cycle in cell motility

How blebs and pseudopods cooperate during chemotaxis

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